Fig. 1. Anatomical contact between the Bolwig nerve and the lateral neurons in wild-type third instar larvae. (A-C) Whole-mounted CNS of a third instar w; GMR-gal4/UAS-gfp larva, with one eye imaginal disc (epifluorescent microscopy). The white box indicates the region shown in greater detail in B and C. Green (A,C): staining of the visual system with GMR-gal4-driven GFP expression. Red (A,B): anti-ß-PDF staining of the lateral neurons. (D,E) Whole-mount of third instar larval CNS (epifluorescent microscopy). The LNs are visualized by anti-ß-PDF antibody (red). Expression of the rhodopsin genes in the Bolwig organ is detected through reporters driven by specific promoters. (D) Green: GFP staining of the BN in a w; UAS-gfp/+; rh5-gal 4/+ larva. (E) Green: ß-galactosidase expression in the BN revealed by anti-ß-gal antibody in a w; rh6-lacZ larva. (F) Confocal reconstruction. Double staining of the BN in a w; rh6-gfp/rh5-lacZ larva shows that rh5 and rh6 are expressed in different axons (and therefore different cells) of the BN. Green, GFP staining of rh6-expressing fibers; red, anti- ß-gal staining of rh5-expressing fibers. No rh1 expression was found in the BN using either rh1-gal4 (with a UAS-gfp reporter) or rh1-norpA (with anti-NORPA antibody) constructs. (G-I) Whole-mounts of third instar larval CNS (confocal microscopy) (G,H, 1 µm single optical section; I, five projected optical sections). (G) Anti- ß-PDF staining (red) reveals the LNs and anti-chaoptin staining (green) reveals the BN in a wild-type larva (w). Like the GMR-gal4 transgene used in A-C, the chaoptin gene is expressed in both larval and adult photoreceptors. (H,I) LNs are visualized by GFP expression (green) in w; UAS-gfp; gal1118 larvae. Anti-ChAT (H) or anti-NORPA (I) immunoreactivities (red) label the BN. No anti-NORPA labeling was observed in the BN of norpA^P24 mutant larvae (not shown). APP, adult photoreceptors projections; BN, Bolwig nerve; DA, dendritic arborization of LNs; DP, dorsal projections of LNs; EID, eye-antenna imaginal disc; LNs, lateral neurons. Arrowheads indicate contact between BN terminus and LNs dendritic arborization. Scale bars: 10 µm.